An increasing number of experiments show a potential role of miRNAs in cancer.Explain

Introduction
An increasing number of experiments show a potential role of miRNAs in cancer. Cancer is a complicated and dynamic disease characterized by abnormal gene expression and cell growth. Cancer has gained significantly enormous attention and efforts due to its high mortality rates. Commonly, scientists research cancer by studying protein-coding genes, which are viewed as the main effectors and controls of oncogenesis. Nonetheless, recent research has shown that non-protein-coding RNAs also play an essential role in tumorigenesis. Among these non-protein-coding RNAs, microRNAs, abbreviated miRNAs, have gained researchers’ attention due to their ability to participate in the regulation of cell proliferation, differentiation, as well as apoptosis. MiRNAs offer an essential aspect of control oncogenesis through gene regulation, and may also function as potential cancer therapy (Slack & Espinosa, 2006).
miRNAs are a class of short non-protein–coding RNAs, comprised of around 22 nucleotides. The first miRNA was characterized in 1993 from the nematode Caenorhabditis elegans. In this organism the lin-4 locus is transcribed into a small RNA, instead of a protein. This RNA molecule is able to inhibit the lin-14 protein expression by directly targeting the 3’ untranslated region of lin-14 mRNA, and thus influences the post-embryonic development of the organism. At the time, scientists thought it was unique to C. elegans, until another small RNA, let-7 that inhibits lin-41 protein expression in C. elegans, was discovered in 2000. The let-7 RNA was then found to be conserved in many other species, leading to the suggestion that let-7 RNA and additional small temporal RNAs might regulate the timing of development in diverse animals, including humans. In 2001, the lin-4 and let-7 RNAs were found to be part of a large class of small RNAs present in C. elegans and human cells. The many RNAs of this class resembled the lin-4 and let-7 RNAs, except their expression patterns were usually inconsistent with a role in regulating the timing of development. This suggested that most RNAs might function in other types of regulatory pathways. At this point, researchers started using the term ‘microRNA’ to refer to this class of small regulatory RNAs. MiRNA molecules are then found in many other species, including plants and animals, playing an essential role in cell development through gene regulation (Iorio & Croce. 2012). Based on the prediction of computational analysis, the human genome contains more than 1,000 miRNAs, among which 300 have already been identified. Estimates illustrate that in the human genome up to 30 percent of the protein-coding genes may be influenced by miRNAs, stressing their significance in gene expression (Slack & Espinosa, 2006). However, the mechanism of how miRNAs act in cancer is not completely figured out.
Since miRNAs influence gene expression, their mechanisms and potential usage in cancer control have been the focus of current scientific study. In order to present a comprehensive awareness of miRNA, this review paper will integrate the results from a number of experiments, analyses, computer modeling, and predictions. This article will focus on six aspects of miRNA’s characteristics: miRNA biogenesis and mechanisms, miRNA expression in human cancer, miRNAs as prognostic and predictive biomarkers for cancer, miRNAs as tumor suppressors, miRNAs as oncogenes, as well as miRNA-based cancer treatment.
miRNA biogenesis and mechanisms
The biogenesis of miRNA starts with the transcription of a large precursor RNA, known as pri-miRNA, by RNA Polymerase II. Commonly, human miRNAs are transcribed from regions in introns of either protein-coding or non-coding transcripts. In the nucleus, the pri-miRNA is processed by a RNase III enzyme, Drosha, and a RNA binding protein DGCR8, resulting in a stem-loop structure, approximately 80-100 nucleotides in length, called pre-miRNA (Denli, Tops, Plasterk, Ketting, & Hannon, 2004). The pre-miRNA is transported from the nucleus into the cytoplasm by exportin 5 (Bohnsack, Czaplinski, & GÖRLICH, 2004), and then processed by another RNase III enzyme, Dicer, releasing a mature double-stranded miRNA duplex. Subsequently, this miRNA duplex binds to Argonaute proteins, leading to the formation of miRNA-Induced Silencing Complex (miRISC), which then regulates the translation of complementary messenger RNA (mRNA).
The mature miRNA mainly recognizes its complementary sequences in the 3′ untranslated region (UTR) of their target mRNAs, typically positions 2–7 in the miRNA. However, recent studies have suggested that miRNA also binds to 5′UTR, often called the “miRNA seed,” or open reading frame (ORF) of the target mRNA. Bioinformatic approaches have taken advantage of this “miRNA seed” to predict miRNA targets across the genome. It has been predicted that a single miRNA can bind over 200 different target transcripts, and, notably, these targets are highly diverse, from transcription factors to transporters. The resulting aberrant miRNA expression may affect a multitude of transcripts, which have profound influence on cancer-related signaling pathways.
miRNA as tumor suppressors
Gain-of-function experiments have suggested the function of a number of dysregulated miRNAs as tumor suppressors. By targeting oncoproteins, these miRNAs disturb a variety of oncogenic pathways, such as CLL and RAS.
miR-15/miR-16 and CLL
The experiment done by Calin et al. is the first research that straight demonstrates the role of dysregulated miRNA in oncogenesis. The study suggests the function of miR-15 and miR-16 as tumor suppressors in CLL. CLL, which is known as chronic lymphocytic leukemia, is a type of cancer that starts from cells that become certain white blood cells in the bone marrow. It is the most common type of leukemia in the Western countries. Except for a chromosome 13q14 deletion in most CCL cases, there are comparatively few chromosome mutations in CLL samples, demonstrating that no gene has been directly linked with CCL. The abnormalities of 13q14 show the oncogenic significance (Calin et al., 2002). After identifying a 31.4kb minimally deleted region at 13q14, Calin et al. successfully found a cluster of two miRNA genes, miR-15 and miR-16, instead of any protein-coding genes, within that deleted region. The further experiments of Calin et al. showed that when compared with normal tissues counterparts, the expression of miR-15 and miR-16 were dramatically reduced in 68% of CLL samples. This study suggests that miR-15 and miR-16 are down-regulated in most CLL patients. Calin’s et al. studies did not reveal any point mutation in CLL.
The expression of miR-15 and miR-16 are significantly reduced, or even totally vanished, in 68% of CLL patients, 50% of mantle cell lymohoma patients, and 60% of prostate cancer patients (Calin et al., 2002). This fact effectively demonstrates the role of miR-15 and miR-16 as tumor suppressors. Although their complete targeting mechanisms are not yet fully recognized, researches have shown that miR-15 and miR-16 suppress tumor probably due to their ability to down-regulate BCL2 protein (Cimmino et al., 2005). BCL2, which stands for B cell lymphoma 2, is a member of regulator proteins that regulate apoptosis. Overexpression of this protein is often found in many kinds of cancer, including CLL. It is classified as an oncogene due to its capability of inhibiting cell death and maintain malignant cell survival. The study done by Cimmino et al. shows a negative correlation between the levels of miR-15 and miR-16 and the expression of BCL2. In normal lymphoid cells, the miR-15 and miR-16 remain high levels and the BCL2 is significantly underexpressed, while in most leukemic cells, both miRNAs present low levels and the BCL2 is overexpressed. These facts, along with further experiments, suggest that BLC2 is a target of miR-15 and miR-16 post-transcriptional repression. Instead of affect mRNA stability, miR-15 and miR-16 interact with the 3’ UTR of BCL2 mRNA straightly, and thus inhibit the expression of BCL2 protein (Cimmino et al., 2005). Furthermore, Cimmino et al. found that BCL2 protein repression by miR-15 and miR-16 triggers apoptosis in a leukemia cell line. Although further studies are needed to better understand the mechanisms of miR-15 and miR-16 processing, and to find any other component that may be potential target of both miRNAs in CLL, the studies done so far illustrate a significant role of miRNA as tumor suppressor.
The let-7 family and Ras
As motioned earlier in the introduction, let-7, along with lin-4, are the first two miRNAs to be discovered. It is the first known human miRNA. Studies performed in nematode Caenorhabditis elegans showed that let-7 regulates the timing of development, which first suggests that miRNAs may play a role in carcinogenesis. According to the research done by Reinhart’s et al., let-7 may serve as temporal switch between larval and adult stages. In wild-type animal seam cells, upregulation of t let-7 is needed to induce cell cycle exit and terminal differentiation at the adult stage. In loss-of-function let-7 mutants, seam cells are unable to exit the cell cycle and fall to differentiate at the proper checkpoints. Instead, they undergo additional division, a hallmark of cancer (Reinhart et al., 2000).
let-7 is found in many species. In addition to C. elegans, it also plays a regulatory role in some other organisms, such as Drosophila and zebrafish (Johnson et al., 2005). In the human genome, the let-7 family comprises 13 members, organized in nine clusters (Boyernas, Park, Hau, Murmann, & Peter, 2010). let-7 is commonly viewed as a tumor suppressor. This is due to its relatively underexpression in many types of cancer, including lung cancer, compare to normal human tissues. Additionally, at least four clusters of let-7 family have been reported to be totally deleted in human cancer. Studies also show that the overexpression of let-7 restrains colony formation, and thus inhibits the growth of a lung adenocarcinomas cell line in vitro (Johnson et al., 2005). Although the mechanisms through which let-7 is able to controls cell cycle exit are not fully understood, these experiments provide us evidence that miRNAs have the capability of suppressing tumor growth.
In the processes of studying let-7, RAS has gained scientists’ concentration. RAS is a family of proteins, participated in cellular signal transduction. It delivers signals from the receptors on the cell surface, and ultimately turns on/off genes involved in cell growth, differentiation, and survival. In many kinds of cancer, the signal transduction pathways are interrupted due to mutations of the Ras genes. When mutated, the RAS proteins are commonly overexpressed compare to normal human tissues, and become permanently activated, resulting in continuously signal transduction and unstopped cell proliferation, which is a key feature of cancer. The experiments performed by Johnson et al. demonstrate the reciprocal expression of RAS and a member of let-7 family, miR-84, in lung tumor cells. let-7 is able to negatively regulation the expression of RAS. If let-7 is overexpressed in human cancer cells, the levels of RAS proteins decreased dramatically. On the contrary, the underexpression of let-7 results in significantly reduced levels of RAS. Further study confirms that let-7 regulates the expression of RAS protein by directly targeting the 3’ UTR of Ras miRNA (Johnson et al., 2005). Since the oncogenic activity RAS protein demonstrates, the let-7 family is viewed as potential cancer therapy for not only lung cancer, but also other tumors associated with the overexpression of RAS.
Other miRNAs as tumor suppressors
Researches have identified other miRNAs serving as tumor inhibitors. For instance, miR-26a is able to restrain hepatocellular carcinoma cell proliferation and induce cancer cell apoptosis by inhibiting the expression of cyclin D2 and E2 (Kota et al., 2009); miR-335 suppresses breast cancer invasion by regulating gene expression and targeting progenitor cell transcription factor SOX4 and extracellular matrix component tenascin C (Tavazoie et al., 2008). Further studies are necessary to better understand the mechanisms through which miRNAs regulate carcinogenesis, and find more efficiently and effectively therapy using miRNAs to treat cancer.
miRNA as oncogenes
A miRNA is viewed as an oncogene when it is notably overexpressed in tumors (Zhang, Pan, Cobb, & aNDERSON, 2007). Commonly, it promotes cancer cell development by downregulating tumor suppressors, and/or downregulating genes controlling cell differentiation and apoptosis. Various miRNAs have been reported that act as oncogenes, while only a minority have been fully characterized.
miR-17-92
miR-17-92 is an example of oncogenes. The miR-17-92 cluster is located at chromosome 13q31, a locus that is amplified in a variety of cancers, including lung cancer and several types of lymphoma, such as diffuse large-B-cell lymphoma, mantle cell lymphoma, and follicular lymphoma. miR-17-92 is encoded by C13orf25, the only gene overexpressed in this amplified locus (Slack & Espinosa, 2006). The study done by Hayashita et al. illustrates that the overexpression of miR-17-92 is dramatically notable in diverse cancer types, such as lung cancer and lymphoma. It is also validated that the miR-17-92 cluster is able to promote lung tumor cell growth. These discoveries obviously suggest that miR-17-92 plays a role in tumorigenesis. However, understanding how miR-17-92 works in cancer development and identification of potential targets of miR-17-92 are still in progress. Bioinformatics tools show that various genes can be targeted by miR-17-92 cluster. For instance, one tumor suppressor gene, PTEN, which stands for phosphatase and tensin homolog that is deleted on chromosome ten and promotes apoptosis, is predicted to be a target of miR-17-92 cluster (Zhang, Pan, Cobb, & aNDERSON, 2007). A recent study performed by Kanzaki et al. has successfully validated one direct target, RAB14 protein, from in total 112 up-regulated proteins that were detected and identified as potential targets of miR-17-92 cluster.
More researches suggest that the expression of miR-17-92 is associated with the expression of Myc gene. MYC is a transcription factor, regulating cell growth by inducing proliferation and apoptosis. In human cancers, Myc is usually mutated, resulting in the permanently expression of MYC. The overexpression of MYC interrupts many other gene expression pathways, which induces unstopped cell proliferation, and eventually leads to the formation of cancer.
Other miRNAs as oncogenes
miR-21 is able to suppress metastasis-related tumor suppressor genes, including tropomyosin 1(TPM1), programmed cell death 4(ODCD4) and maspin (Zhu, Wu, Wu, Nie, Sheng, & Mo, 2008).
miRNA-based cancer treatment
Applying RNA-based treatment for cancer remains elusive for medical practitioners and in the general medical field. Although the studies of miRNAs, especially how miRNAs act in cancer, is not fully understood, miRNAs have already gained enough concentration in terms of function as promising cancer therapeutic agents. This is due to miRNAs’ diversity and the role they play in the regulation of gene expression in unicellular and multicellular eukaryotes. Apart from that, some miRNAs have the capacity to regulate a good number of transcripts thereby making it possible to coordinate sophisticated gene expression programs. However, no clinical or toxicological studies are published yet. The miRNA-based cancer treatment is still at preclinical stage.
A potential miRNA-based cancer treatment focuses on the miRNAs that are significantly underexpressed in carcinogenesis, for instance, miR-15/miR-16 and the let-7 family mentioned earlier. In this case, scientists deliver such tumor-targeting miRNAs into cancer cells in order to counterbalance the shortage and inhibit tumor development. In the study performed by Chen et al., they developed a liposome-polycation-hyaluronic acid (LPH) nanoparticles and then modified it with single-chain antibody fragment (scFv), a tumor targeting antibody with high affinity and low antigenicity that would help in delivering miRNAs into selected murine B16F10 melanoma cells. The delivery of miR-34a, which was found to hinder survivin expression, suppress MAPK pathway, and stimulate apoptosis in B16F10 cells, dramatically inhibited the surviving expression and diminished tumor burden. Interestingly, they detected an improved antitumor effect when miR-34a is co-formulated with siRNAs in the nanoparticles (Chen, Zhu, Zhang, Liu, & Huang, 2010).
Studies done by Kota et al. also suggest that miRNAs had the potential of being used in the treatment against cancer because of their ability to manipulate cellular behavior.
In their experiment on liver cancer cells in vitro, they found that the expression of miR-26a, which is underexpressed in hepatocellular carcinoma (HCC) cells, leads to cell cycle arrest by directly targeting cyclins D2 and E2. In their attempt to deliver miR-26a into murine HCC cells through adeno-associated virus (AVV), this miRNA was found to protect the body from the progression of disease, induce tumor-particular apoptosis, and inhibit proliferation of cancerous cells (Kota et al, 2009). Nevertheless, although studies suggests that miRNAs might be suitably applied as a therapeutic agent in the treatment against cancer, side effects of therapeutic miRNA delivery are still a challenge: the virus using as vector may integrate its genetic material into the host chromosome, leading to unpredictable mutation; adeno-associated virus is possible to induce immune response when enter host cells; the delivered miRNAs are unable to distinguish between normal and tumor cells (Iorio & Croce, 2012).
In addition to delivery cancer suppressor miRNAs that are underexpressed in tumor, another potential strategy miRNA-based cancer treatment can use is inhibiting the overexpression of oncogenic miRNAs. This can be done by designing chemically modified anti-miRNA oligonucleotides (AMOs). A few AMOs under distinct modifications are developed, with the intention of making them less toxic, more specific, more stable, and have higher binding affinity to RNA. The most common one is 2’-O-methylated oligonucleotides, abbreviated 2’-O-Me AMOs. The study done by Esau et al. shows that the delivery of 2’-O-Me AMOs successfully inhibits the expression of miR-122 in mice (Iorio & Croce, 2012). Another prevailing AMOs is the locked nucleic acid (LNA)-modified AMOs, which has significantly increased affinity for RNA (Espinosa & Slack, 2006). It is validated that miR-122 is also underexpressed in mice when LNA-modified AMOs are introduced.
Discovering safe and effective methods to deliver miRNAs or AMOs into tumor cells is still a challenge. Until now no clinical or toxicological achievements have been reported. As scientists getting better understanding of miRNAs, all these might successfully achieve their promise as therapeutic agents for cancer.